Efficient cell culture system for hepatitis C virus genotype 2B

ABSTRACT

The present inventors developed hepatitis C virus 2b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 2b reference strain J8. Sequence analysis of recovered 2b/2a recombinants from 2 transfection experiments revealed that 2b/2a was genetically stable. Conclusion: The developed 2b/2a viruses provide a robust in vitro tool for research in HCV genotype 2b, including vaccine studies and functional analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage application of International Patent Application No PCT/DK2009/050189, filed Jul. 24, 2009, which is incorporated herein by reference in its entirety and which claims the benefit of European Application No. EP 08162466.0, filed Aug. 15, 2008.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing named “66146_(—)94676_SEQ_LST.txt” and which is 81153 bytes in size, is electronically filed herewith and herein incorporated by reference in its entirety. This Sequence Listing consists of SEQ ID NOs: 1-14.

FIELD OF THE INVENTION

The present invention provides infectious recombinant hepatitis C genotype 2 viruses (HCV), and vectors, cells and animals comprising the same. The present invention provides methods of producing the infectious recombinant HCV genotype 2, and their use in identifying anti-HCV therapeutics including use in vaccines and diagnostics, as well as sequences of HCV associated with HCV pathogenesis.

BACKGROUND

Hepatitis C is one of the most widespread infectious diseases in the world. About 180 million people are infected with hepatitis C virus (HCV) worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.

HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and post-translationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.

HCV isolates from around the world exhibit significant genetic heterogeneity. At least 6 major HCV genotypes (genotypes 1-6) have been identified, which differ by 31-33% at the nucleotide level and deduced amino acid level. In addition, there are numerous subtypes (a, b, c, etc.), which differ by 20-25% on the nucleotide and deduced amino acid level.

While HCV genotypes 1-3 predominate in the Western World, genotypes 4-6 are more common in areas with high prevalence or even endemic levels of HCV infection. Subtypes 1b and 2b have a significant prevalence in the Western world.

While the only approved treatment for chronic HCV infection, combination therapy with interferon-α and ribavirin, leads to a sustained virologic response in most of genotype 2 or 3 patients, viral clearance is only obtained for about half of patients with genotype 1 or 4. There is no vaccine against HCV.

Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines.

In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).

A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells (Wakita et al., 2005) (Zhong et al., 2005).

At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (C, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo.

Despite the importance of the described cell culture systems they represent only a single subtype (genotype 2a) of HCV. It is important to develop cell culture systems for representative strains of other HCV genotypes, since neutralizing antibodies are not expected to cross-neutralize all genotypes and new specific antiviral compounds might have differential efficiencies against different genotypes. For the genotype specific study of the function of the structural proteins, p7 and NS2 as well as related therapeutics such as neutralizing antibodies, fusion inhibitors, ion-channel blockers and protease inhibitors, it would be sufficient to construct intergenotypic recombinant viruses in analogy to J6/JFH.

Pietschmann et al. 2006 disclose construction and characterization of infectious intra- and intergenotypic hepatitis C virus recombinants. The authors created a series of recombinant genomes allowing production of infectious genotype 1a, 1b, 2a and 3a particles by constructing intra- and intergenotypic recombinant genomes between the JFH1 isolate and the HCV isolates: H77 (genotype 1a), Con1 (genotype 1b), J6 (genotype 2a) and 452 (genotype 3a) respectively. Thus, disclosing genotype subtypes completely different from the genotype disclosed in the present application.

The infectious titers of the 1a, 1b and 3a genotypes disclosed in Pietschmann et al. 2006 are not at a level sufficiently high for practical utilization in functional analysis, drug and vaccine development or other applications. For such applications, including screening of potential drugs and development of potential vaccine candidates, the skilled person will know that infectivity titers below 10³ TCID50/mL contain insufficient amounts of infectious virus.

Accordingly, the study does not attempt cell culture adaptation of the genotype recombinants, e.g. by serial passage of cell culture derived viruses to naïve cells and it is not investigated whether adaptive mutations develop after transfection in cell culture. In fact, Pietschmann et al does not even provide any sequence data of the virus produced in the cell culture.

SUMMARY OF INVENTION

In this study, the present inventors used the J8 reference isolates (genotype 2b) to construct a viable, JFH1-based genome. The present inventors passaged J8/JFH1 virus in cell culture and obtained both high infectivity titers, high HCV RNA titers and identified one putative adaptive mutation.

The present inventors have developed robust cell culture systems for HCV genotype 2b. This is an important advance for the study of HCV, since it permits detailed molecular studies of HCV and enhances the potential for developing broadly reactive reagents against HCV, including but not limited to small molecule drugs, antibodies and vaccines. Accordingly, the present invention may be used for individualised treatment of patients infected with genotype 2b.

In one aspect the present invention relates to a replicating RNA comprising the structural genes (Core, E1, E2), p7 and the non-structural gene NS2 of genotype 2b and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B from the JFH1 strain.

In another aspect the present invention pertains to an isolated nucleic acid molecule which encodes human hepatitis C virus of genotype 2b/JFH1, wherein said molecule is capable of expressing said virus when transfected into cells.

In yet another aspect the present invention pertains to a composition comprising a nucleic acid molecule according to the present invention, a cassette vector for cloning viral genomes, methods for producing a cell which replicates HCV 2b/JFH1 RNA and cells obtainable there from.

In another aspect the present invention pertains to methods for producing a hepatitis C virus particle, methods for in vitro producing a hepatitis C virus-infected cell.

In a further aspect the present invention pertains to methods for screening an anti-hepatitis C virus substance, hepatitis C vaccines comprising a hepatitis C virus particle, methods for producing a hepatitis C virus vaccine and antibodies against hepatitis C virus.

DETAILED DESCRIPTION

The present invention advantageously provides hepatitis C virus (HCV) nucleotide sequences capable of replication, expression of functional HCV proteins, and infection in vivo and in vitro for development of antiviral therapeutics and diagnostics.

Nucleic Acid Molecules (cDNA Clones and RNA Transcripts)

In a broad aspect, the present invention is directed to a genetically engineered hepatitis C virus (HCV) encoded by nucleic acid sequences such as a complementary DNA (cDNA) sequence and replicating RNA (J8/JFH1) comprising the structural genes (Core, E1, E2), p7 and the non-structural gene NS2 of genotype 2b (e.g. strain J8) and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B from the JFH1 strain (genotype 2a, genbank accession number AB047639).

Thus in one embodiment, the present invention relates to a replicating RNA comprising the structural genes (Core, E1, E2), p7 and the non-structural gene NS2 of genotype 2b and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B from the JFH1 strain.

In another embodiment the genotype 2b is of the strain J8.

The invention provides an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, which nucleic acids comprise an intergenotypic HCV genome. In one embodiment, the intergenotypic HCV genome comprises sequences encoding structural genes (Core, E1, E2), p7 and nonstructural genes (NS2) from a first HCV strain, and sequences encoding the 5′ untranslated region (UTR), nonstructural genes NS3, NS4A, NS4B, NS5A, NS5B, and the 3′ UTR from a second HCV strain.

In one embodiment, the first HCV strain and the second HCV strain are from different genotypes.

In one embodiment, the first HCV strain is strain J8, and in another embodiment, the second HCV strain is strain JFH1.

In one embodiment, the HCV nucleic acid molecule of the present invention comprises the nucleic acid sequence (cDNA) of J8/JFH1, SEQ ID NO: 1. In another embodiment the nucleic acid molecule has at least a functional portion of a sequence as shown in SEQ ID NO: 1 which represents a specific embodiment of the present invention exemplified herein.

In yet an embodiment the nucleic acid molecule comprises the nucleic acid sequence with a sequence identity of at least 90% to that of SEQ ID NO: 1.

In another embodiment, the nucleic acid comprises a sequence sharing at least 90% identity with that set forth in SEQ ID NO: 1, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity.

As commonly defined “identity” is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. Thus, in the present context “sequence identity” is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)×100). In one embodiment the two sequences are the same length.

One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. “scoring matrix” and “gap penalty” may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

It should be noted that while SEQ ID NO: 1 is a DNA sequence, the present invention contemplates the corresponding RNA sequence, and DNA and RNA complementary sequences as well.

In a further embodiment, a region from an HCV isolate is substituted for a corresponding region, e.g., of an HCV nucleic acid having a sequence of SEQ ID NO: 1.

In another embodiment, the HCV nucleic acid is a DNA that codes on expression or after in vitro transcription for a replication-competent HCV RNA genome, or is itself a replication-competent HCV RNA genome.

In one embodiment, the HCV nucleic acid of the invention has a full-length sequence as depicted in or corresponding to SEQ ID NO: 1. Various modifications for example of the 5′ and 3′ UTR are also contemplated by the invention. In another embodiment, the nucleic acid further comprises a reporter gene, which, in one embodiment, is a gene encoding neomycin phosphotransferase, Renilla luciferase, secreted alkaline phosphatase (SEAP), Gaussia luciferase or the green fluorescent protein.

Naturally, as noted above, the HCV nucleic acid sequence of the invention is selected from the group consisting of double stranded DNA, positive-sense cDNA, or negative-sense cDNA, or positive-sense RNA or negative-sense RNA or double stranded RNA. Thus, where particular sequences of nucleic acids of the invention are set forth, both DNA and corresponding RNA are intended, including positive and negative strands thereof.

In a further embodiment, the nucleic acid sequence of SEQ ID NO: 1 or the said nucleic acid sequence with or without any mutation described in this document is obtained by any other means than what is described above.

In another embodiment, the complementary DNA (cDNA) provided by the present invention encodes human hepatitis C virus of genotype 2b/JFH1, wherein said molecule is capable of expressing said virus when transfected into cells and further capable of infectivity in vivo and wherein said molecule encodes the amino acid sequence of J8/JFH1, SEQ ID NO: 2.

According to various aspects of the invention, HCV nucleic acid, including the polyprotein coding region, can be mutated or engineered to produce variants or derivatives with, e.g., silent mutations, conservative mutations, etc. In a further preferred aspect, silent nucleotide changes in the polyprotein coding regions (i.e., variations of the first, second or third base of a codon leading to a new codon that encodes the same amino acid) are incorporated as markers of specific HCV clones.

Thus, one aspect of the present invention relates to any of the amino acid sequences disclosed herein, such as but not limited to SEQ ID NO: 2.

In yet an embodiment the isolated nucleic acid molecule encodes the amino acid sequence with a sequence identity of at least 90% to that of SEQ ID NO: 2.

In another embodiment, the amino acid sequences comprises a sequence sharing at least 90% identity with that set forth in SEQ ID NO: 2, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity.

It is to be understood that a sequence identity of at least 90%, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity applies to all sequences disclosed in the present application.

Nucleic acid molecules according to the present invention may be inserted in a plasmid vector for translation of the corresponding HCV RNA. Thus, the HCV DNA may comprise a promoter 5′ of the 5′-UTR on positive-sense DNA, whereby transcription of template DNA from the promoter produces replication-competent RNA. The promoter can be selected from the group consisting of a eukaryotic promoter, yeast promoter, plant promoter, bacterial promoter, or viral promoter.

In one embodiment the present invention provides a cassette vector for cloning viral genomes, comprising, inserted therein, the nucleic acid sequence according to the invention and having an active promoter upstream thereof.

Adaptive Mutations

Adapted mutants of a HCV-cDNA construct or HCV-RNA full-length genome with improved abilities to generate infectious viral particles in cell culture compared to the original HCV-cDNA construct or the original HCV-RNA full-length genome are characterized in that they are obtainable by a method in which the type and number of mutations in a cell culture adapted HCV-RNA genome are determined through sequence analysis and sequence comparison and these mutations are introduced into a HCV-cDNA construct, particularly a HCV-cDNA construct according to the present invention, or into an (isolated) HCV-RNA full-length genome, either by site-directed mutagenesis, or by exchange of DNA fragments containing the relevant mutations.

In contrast to JFH1-based intergenotypic recombinants of genotypes 1a, 1b, 3a, 4a, 5a and 6a (Gottwein et al. 2007, Scheel et al. 2008, Jensen et al. in press, Gottwein et al. unpublished results), viability and efficient growth of J8/JFH1 apparently does not depend on adaptive mutations. The present inventors here report one putative adaptive mutation, which might be advantageous but apparently not necessary for efficient formation and release of viral particles in cell culture, and thus the present invention relates to this adaptive mutations in the present use as well as use in other strains by changing equivalent positions of such genomes to the adapted nucleotide or amino acid described. Efficient growth of J8/JFH1 without adaptive mutations is indeed surprising, however, could be explained by the fact that J8/JFH1 is a recombinant of two different HCV subtypes (2b and 2a) and not two different genotypes. HCV RNA and HCV infectivity titers observed in Huh7.5 cell cultures infected with J8/JFH1 viruses (Table 3 and Table 4) were comparable to those observed in cultures infected with the reference virus J6/JFH1 as well as other JFH1-based intergenotypic recombinants (e.g. genotypes 1a and 6a) developed by the present investigators. However, viability and efficient growth of intergenotypic recombinants of genotypes 1a, 1b, 3a, 4a, 5a and 6a was dependent on adaptive mutations.

The preferred HCV-cDNA construct, HCV-RNA full-length genome with the ability to release viral particles in cell culture, which is consequently highly suitable for practical use, is characterized in that it contains none or the following nucleic acid exchange and/or none or the following amino acid exchange.

In two independent RNA transfections of Huh7.5 cells, J8/JFH1 spread comparably to J6/JFH with approximately 20% and 80% HCV antigen positive cells on days 1 and 4, respectively (FIG. 1A). The supernatant infectivity titers increased from ˜10^(1.5) TCID₅₀/mL on day 1 to ˜10^(3.5) TCID₅₀/mL on day 4 (FIG. 1B). During viral passage (FIG. 1C), HCV RNA and infectivity titers were ˜10⁷ IU/mL (FIG. 1D) and ˜10^(4.5) TCID₅₀/mL (data not shown; see also Table 3 and 4), respectively. Analysis of recovered viruses (days 10 and 13 post-infection, respectively) showed that J8/JFH1 was genetically stable in two first passages performed from the first transfection (data not shown and exp. 1 in FIGS. 1C and D). In the passage of virus from the second transfection (exp. 2 in FIGS. 1C and D), G4458A (R1373Q) was present as a 50/50 quasispecies with the original sequence (virus from day 13 post-infection).

One embodiment of the present invention relates to adaptive mutations, wherein the adaptive mutation is a mutation that can be observed by clonal or direct sequencing of recovered replicating genomes of SEQ ID NO: 1.

Thus in a further embodiment, the present invention relates to nucleic acid molecules according to the present invention, wherein said molecule comprises none or one adaptive mutations in NS3.

In another embodiment the present invention relates to an isolated nucleic acid molecule which encodes human hepatitis C virus of genotype 2b/JFH1, wherein said molecule is capable of expressing said virus when transfected into cells and wherein said molecule encodes the nucleic acid sequence with a sequence identity of at least 90% to that of SEQ ID NO: 1 and wherein the genotype 2b is strain J8.

In yet an embodiment the present invention relates to an isolated nucleic acid molecule which encodes human hepatitis C virus of genotype 2b/JFH1, wherein said molecule is capable of expressing said virus when transfected into cells and wherein said molecule encodes the nucleic acid sequence with a sequence identity of at least 90% to that of SEQ ID NO: 2 and wherein the genotype 2b is strain J8.

In the context of the present invention the term “adaptive mutation” is meant to cover mutations identified in passaged J8/JFH1 viruses that provide the original J8/JFH1 and any other HCV sequence the ability to grow efficiently in culture. Furthermore all introductions of mutations into the J8/JFH1 sequences described, whether or not yielding better growth abilities, and the introduction of these mutations into any HCV sequence should be considered.

Thus the described mutation enable the HCV-RNA genome (e.g. derived from a HCV-cDNA clone) to form viral particles in and release these from suitable cell lines. In addition the described mutation might change the function of NS3 in favourable ways, which might be exploited in other experimental systems employing these proteins. This also includes other HCV genomes with adaptive mutations, all of them, combinations of them or individual mutations that grow in culture. In this case the titers might be lower than those listed.

One embodiment of the present invention relates to adaptive mutations, wherein said adaptive mutation is a replacement of G in position 4458 of SEQ ID NO: 1 with A as represented in SEQ ID NO: 13.

In another embodiment all the amino acid change observed herein is provided by the present application. The skilled addressee can easily obtain the same amino acid change by mutating another base of the codon and hence all means of obtaining the given amino acid sequence is intended.

An embodiment of the present invention relates to adaptive mutations, wherein said adaptive mutation is a replacement of R in position 1373 of SEQ ID NO: 2 with Q as represented in SEQ ID NO: 14.

Titer

To determine the efficiency of the developed system, HCV RNA titers are determined in IU/ml (international units/ml) with Taq-Man Real-Time-PCR and infectious titers are determined with a 50% tissue culture infectious dose method. This titer shows the dilution of the examined viral stock, at which 50% of the replicate cell cultures used in the essay become infected and is given in TCID50/ml. Alternatively the infectious titers are determined as FFU/ml (focus forming unites/ml) (Table 3 and 4); in this method, infectivity titers are determined by infection of cell culture replicates with serial dilutions of virus containing supernatants and, following immuno-stainings for HCV antigens, counting of HCV-antigen positive cell foci.

HCV RNA titers and infectivity titers can be determined extracellularly, in cell culture supernatant (given as IU and TCID₅₀ or FFU per ml, respectively) or intracellularly, in lysates of pelleted cells (given as IU and TCID₅₀ or FFU related to a given cell number, which was lysed) (Table 4).

One embodiment of the present invention relates to a nucleic acid molecule of the present invention, wherein said molecule is capable of generating a HCV RNA titer of 10⁴ IU/ml or above following transfection and/or subsequent viral passage, such as a titer of at least 10⁵ IU/mL, such as a titer of at least 10⁶ IU/mL, such as a titer of at least 10⁷ IU/mL, such as a titer of at least 10⁸ IU/mL, such as a titer of at least 10⁹ IU/mL, such as a titer of at least 10¹⁰ IU/mL, such as a titer of at least 10¹¹ IU/mL, or such as a titer of at least 10¹² IU/mL.

In another embodiment, the present invention relates to a nucleic acid molecule according to the invention, wherein said molecule is capable of generating a HCV infectivity titer of at least 10² TCID₅₀/ml or above following transfection and/or subsequent viral passage, such as a titer of at least 10³ TCID50/ml, such as a titer of at least 10⁴ TCID₅₀/ml, such as a titer of at least 10⁵ TCID₅₀/ml, such as a titer of at least 10⁶ TCID₅₀/ml, such as a titer of at least 10⁷ TCID₅₀/ml, such as a titer of at least 10⁸ TCID₅₀/ml, such as a titer of at least 10⁹ TCID₅₀/ml or such as a titer of at least 10¹⁰ TCID₅₀/ml.

It is of course evident to the skilled addressee that the titers described here are obtained using the assay described in this text. Any similar or equivalent titer determined by any method is thus evidently within the scope of the present invention.

Compositions

One embodiment of the present invention relates to a composition comprising a nucleic acid molecule according to the invention suspended in a suitable amount of a pharmaceutical acceptable diluent or excipient.

In another embodiment, this invention provides for compositions comprising an isolated nucleic acid, vector or cell of this invention, or an isolated nucleic acid obtained via the methods of this invention.

In one embodiment, the term “composition” refers to any such composition suitable for administration to a subject, and such compositions may comprise a pharmaceutically acceptable carrier or diluent, for any of the indications or modes of administration as described. The active materials in the compositions of this invention can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

It is to be understood that any applicable drug delivery system may be used with the compositions and/or agents/vectors/cells/nucleic acids of this invention, for administration to a subject, and is to be considered as part of this invention.

The compositions of the invention can be administered as conventional HCV therapeutics. The compositions of the invention may include more than one active ingredient which interrupts or otherwise alters groove formation, or occupancy by RNA or other cellular host factors, in one embodiment, or replicase components, in another embodiment, or zinc incorporation, in another embodiment.

The precise formulations and modes of administration of the compositions of the invention will depend on the nature of the anti-HCV agent, the condition of the subject, and the judgment of the practitioner. Design of such administration and formulation is routine optimization generally carried out without difficulty by the practitioner.

It is to be understood that any of the methods of this invention, whereby a nucleic acid, vector or cell of this invention is used, may also employ a composition comprising the same as herein described, and is to be considered as part of this invention.

“Pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term “excipient” refers to a diluent, adjuvant, carrier, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response. Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvmm. Preferably, the adjuvant is pharmaceutically acceptable.

Cells

The nucleotides of the present invention may be used to provide a method for identifying additional cell lines that are permissive for infection with HCV, comprising contacting (e.g. transfecting) a cell line in tissue culture with an infectious amount of HCV RNA of the present invention, e.g., as produced from the plasmid clones, and detecting replication and formation and release of viral particles of HCV in cells of the cell line.

Naturally, the invention extends as well to a method for identifying an animal that is permissive for infection with HCV, comprising introducing an infectious amount of the HCV RNA, e.g., as produced by the plasmids, to the animal, and detecting replication and formation and release of viral particles of HCV in the animal. By providing infectious HCV, e.g. comprising a dominant selectable marker, the invention further provides a method for selecting for HCV with further adaptive mutations that permit higher levels of HCV replication in a permissive cell line or animal comprising contacting (e.g. transfecting) a cell line in culture, or introducing into an animal, an infectious amount of the HCV RNA, and detecting progressively increasing levels of HCV RNA and infectious HCV viral particles in the cell line or the animal.

In a specific embodiment, the adaptive mutation permits modification of HCV tropism. An immediate implication of this aspect of the invention is creation of new valid cell culture and animal models for HCV infection.

The permissive cell lines or animals that are identified using the nucleic acids of the invention are very useful, inter alia, for studying the natural history of HCV infection, isolating functional components of HCV, and for sensitive, fast diagnostic applications, in addition to producing authentic HCV virus or components thereof.

Because the HCV DNA, e.g., plasmid vectors, of the invention encode HCV components, expression of such vectors in a host cell line transfected, transformed, or transduced with the HCV DNA can be effected.

For example, a baculovirus or plant expression system can be used to express HCV virus particles or components thereof. Thus, a host cell line may be selected from the group consisting of a bacterial cell, a yeast cell, a plant cell, an insect cell, and a mammalian cell.

In one embodiment, the cell is a hepatocyte, or in another embodiment, the cell is the Huh-7 hepatoma cell line or a derived cell line such as Huh7.5, Huh7.5.1 cell line.

In one embodiment, the cell, or in another embodiment, cell systems of this invention comprise primary cultures or other, also non hepatic cell lines. “Primary cultures” refers, in one embodiment, to a culture of cells that is directly derived from cells or tissues from an individual, as well as cells derived by passage from these cells, or immortalized cells.

In one embodiment, “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. The term “cell lines” also includes immortalized cells. Often, cell lines are clonal populations derived from a single progenitor cell. Such cell lines are also termed “cell clones”. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell clones referred to may not be precisely identical to the ancestral cells or cultures. According to the present invention, such cell clones may be capable of supporting replication of a vector, virus, viral particle, etc., of this invention, without a significant decrease in their growth properties, and are to be considered as part of this invention.

It is to be understood that any cell of any organism that is susceptible to infection by or propagation of an HCV construct, virus or viral particle of this invention is to be considered as part of this invention, and may be used in any method of this invention, such as for screening or other assays, as described herein.

Thus in one embodiment the present invention relates to a method for producing a cell which replicates HCV 2b/JFH1 RNA and produces a virus particle comprising introducing the said RNA according to the invention into a cell.

In one embodiment the present invention relates to a method for producing a cell, which replicates an RNA comprising the structural genes (Core, E1, E2), p7 and the non-structural gene NS2 of genotype 2b, strain J8, and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B from the JFH1 strain and produces a virus particle comprising introducing the said RNA into a cell wherein said RNA encodes an amino acid sequence comprising one adaptive mutation, said adaptive mutation is a replacement of R at position 1373 of SEQ ID NO: 2 with Q.

In one embodiment the 2b strain is J8.

Also, a method for in vitro producing a hepatitis C virus-infected cell comprising culturing the cell which produces virus particles of the present invention and infecting other cells with the produced virus particle in the culture.

Naturally, the invention extends to any cell obtainable by such methods, for example any in vitro cell line infected with HCV, wherein the HCV has a genomic RNA sequence as described herein. Such as a hepatitis C virus infected cell obtainable by any of the methods described.

In one embodiment, the cell line is a hepatocyte cell line such as Huh7 or derived cell lines e.g. Huh7.5 or Huh7.5.1.

In another embodiment the cell is Huh7.5.

In another embodiment the cell is any cell expressing the genes necessary for HCV infection and replication, such as but not limited to CD81, SR-BI, Claudin-1, -4, -6 or -9 and the low-density lipid receptor.

Importance of CD81 for HCV entry has in previous studies been shown for pseudoviral particles (HCVpp) of genotypes 1-6, and for cell culture derived HCV (HCVcc) of genotypes 1a (H77), 1b (Con-1), 3a (S52), 4a (ED43) and 5a (SA13). Blocking of SR-BI receptors was found to inhibit infection with HCVpp of genotypes 1-6. In the HCVcc system, genotypes 2a and 5a in previous studies depended on SR-BI. In comparative studies, the present investigators showed that entry of genotypes 1, 2 and 6 was efficiently inhibited when relative high doses of blocking antibodies against the respective HCV co-receptor were used (FIG. 4). Thus, CD81 and SR-BI play an important role for entry of prototype isolates of the six major genotypes and important subtypes 1b and 2b. Future studies will be required to determine if the different levels of inhibition seen at lower antibody doses are due to stochastical effects or indicate different modes of entry.

The invention further provides various methods for producing HCV virus particles, including by isolating HCV virus particles from the HCV-infected non-human animal of invention; culturing a cell line of the invention under conditions that permit HCV replication and virus particle formation; or culturing a host expression cell line transfected with HCV DNA under conditions that permit expression of HCV particle proteins; and isolating HCV particles or particle proteins from the cell culture. The present invention extends to an HCV virus particle comprising a replication-competent HCV genome RNA, or a replication-defective HCV genome RNA, corresponding to an HCV nucleic acid of the invention as well.

Virus Particle

The production of authentic virus proteins (antigens) may be used for the development and/or evaluation of diagnostics. The cell culture system according to the invention also allows the expression of HCV antigens in cell cultures. In principle these antigens can be used as the basis for diagnostic detection methods.

The production of HCV viruses and virus-like particles, in particular for the development or production of therapeutics and vaccines as well as for diagnostic purposes is an embodiment of the present invention. Especially cell culture adapted complete HCV genomes, which could be produced by using the cell culture system according to the invention, are able to replicate and form viral particles in cell culture with high efficiency. These genomes have the complete functions of HCV and in consequence they are able to produce infectious viruses.

Thus in one embodiment the present invention relates to a method for producing a hepatitis C virus particle of the present invention or parts thereof, comprising culturing a cell or an animal to allow either to produce the virus.

In another embodiment the inventions provides a hepatitis C virus particle obtainable by the method described.

Because the invention provides, inter alia, infectious HCV RNA, the invention provides a method for infecting an animal with HCV which comprises administering an infectious dose of HCV RNA, such as the HCV RNA transcribed from the plasmids described above, to the animal. Naturally, the invention provides a non-human animal infected with HCV of the invention, which non-human animal can be prepared by the foregoing methods.

A further advantage of the present invention is that, by providing a complete functional HCV genome, authentic HCV viral particles or components thereof, which may be produced with native HCV proteins or RNA in a way that is not possible in subunit expression systems, can be prepared.

In addition, since each component of HCV of the invention is functional (thus yielding the authentic HCV), any specific HCV component is an authentic component, i.e., lacking any errors that may, at least in part, affect the clones of the prior art. Indeed, a further advantage of the invention is the ability to generate HCV virus particles or virus particle proteins that are structurally identical to or closely related to natural HCV virions or proteins. Thus, in a further embodiment, the invention provides a method for propagating HCV in vitro comprising culturing a cell line contacted with an infectious amount of HCV RNA of the invention, e.g., HCV RNA translated from the plasmids described above, under conditions that permit replication of the HCV RNA.

Further the viability of the developed viruses may be determined in vivo, either in SCID-uPA mice engrafted with human liver tissue or in chimpanzees as shown in Lindenbach et al. 2006.

In one embodiment, the method further comprises isolating infectious HCV. In another embodiment, the method further comprises freezing aliquots of said infectious HCV. According to this aspect of the invention, and in one embodiment, the HCV is infectious following thawing of said aliquots, and in another embodiment, the HCV is infectious following repeated freeze-thaw cycles of said aliquots.

Screening for anti-viral drugs and the determination of drug resistance.

It can be assumed that resistance to therapy occurs due to the high mutation rate of the HCV genome. This resistance, which is very important for the clinical approval of a substance, can be detected with the cell culture system according to the invention. Cell lines, in which the HCV-RNA construct or the HCV genome or subgenome replicates and produces infectious viral particles, are incubated with increasing concentrations of the relevant substance and the replication of the viral RNA is either determined by means of an introduced reporter gene or through the qualitative or quantitative detection of the viral nucleic acids or proteins. The release of viral particles is determined by measuring HCV RNA and infectivity titers in the cell culture supernatant. Resistance is given if no or a reduced inhibition of the replication and release of viral particles can be observed with the normal concentration of the active substance. The nucleotide and amino acid replacements responsible for the therapy resistance can be determined by recloning the HCV-RNA (for example by the means of RT-PCR) and sequence analysis. By cloning the relevant replacement(s) into the original construct its causality for the resistance to therapy can be proven.

While the replicon systems facilitated testing of drugs interfering with replication such as NS3/4A protease and polymerase inhibitors, the variant genomes obtained in the present invention may prove useful for different research topics. Genomes with the original J8 Core could be applied to examine genotype 2b specific features of Core.

The systems developed in this invention are ideal candidates for genotype 2b specific testing of therapeutics in general and therapeutics targeting viral entry, assembly and release. Genomes with the J8 sequences are valuable for testing of neutralizing antibodies and other drugs acting on entry level, such as fusion inhibitors.

The present inventors conducted cross-genotype neutralization studies in HCV cell culture systems recapitulating the entire viral life cycle using JFH1-based viruses with envelope sequences of all 6 major genotypes and the important subtypes 1b and 2b, which has previously not been possible (Table 2). HCV E1/E2 assembled on HCV pseudo particles (HCVpp), used in previous neutralization studies could show an unphysiological confirmation, glycosylation pattern and/or lipoprotein association due to the nature of the HCVpp as well as the non-hepatic producer cell-lines used in such experiments.

In such studies the viral particles are incubated with the neutralizing substance, e.g. patient derived antibodies present in serum, prior to incubation with cells permissive and susceptible to viral infection. The neutralizing effect, i.e. the inhibitory effect on viral entry, is measured e.g. by relating the number of focus forming units (FFUs, defined as foci of adjacent infected cells) to the equivalent count in a control experiment done under same circumstances without the active inhibitor molecule.

The inventors of the present invention showed that JFH1-based viruses of the genotype 1a, 1b, 2b, 4a, 5a, 6a and 7a were efficiently neutralized by chronic phase H06 genotype 1a serum derived from reference Patient H (Table 2). The results in the cell culture systems compare well to neutralization experiments using Patient H serum from year 26 (H03) carried out in HCVpp systems with envelope proteins of the same prototype isolates of all 6 HCV genotypes as used in the present application, and heterogeneity between the genotypes is thus as previously reported by Meunier et al. 2005.

In addition the present inventors found that cross-genotype neutralization extended to a chronic phase genotype 4a serum (AA), which efficiently neutralized genotype 2b, 4a, 5a, 6a and 7a (Table 2). Also, the cross-genotype neutralization extended to a chronic phase genotype 5a serum (SA3), which efficiently neutralized genotype 2b, 4a, 5a, 6a and 7a (Table 2). It is of note that genotypes subtypes 2a and 2b, which belong to the same genotype and genotypes 2b and 7a, which have, for isolates of different major genotypes, a relatively high sequence homology, differ in their susceptibility to neutralization.

Accordingly, the JFH1-based cell culture systems which have been developed for HCV genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a and 7a provide a valuable tool for efficiently screening for and identifying new candidate HCV genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a and 7a inhibitors e.g. of entry e.g. in serum derived from infected patients. Accordingly this invention, allows identification and raise of cross-neutralizing antibodies, which is important for the development of active and passive immunization strategies. Furthermore the availability of cell culture grown HCV particles bearing envelope proteins of the six major genotypes enables the development of inactivated whole virus vaccines and comprehensive virus neutralization studies.

In one embodiment the present invention relates to a method for identifying neutralizing antibodies.

In another embodiment the present invention relates to a method for identifying cross-genotype neutralizing antibodies.

In one embodiment the present invention relates to a method of raising neutralizing antibodies.

In another embodiment the present invention relates to a method of raising cross-neutralizing antibodies.

In one embodiment the present invention related to a method for screening new HCV genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a and/or 7a inhibitors or neutralizing antibodies, comprising

-   -   a) culturing at least one selected from the group consisting of         a cell according to the present invention, a hepatitis C virus         infected cell according to the present invention and a hepatitis         C virus particle obtainable by the present invention together         with a hepatitis C virus permissive cell, and     -   b) subjecting said virus or virus infected cell culture to a         blood sample or derivatives thereof from a HCV genotype 1a, 1b,         2a, 2b, 3a, 4a, 5a, 6a and/or 7a infected patient     -   c) detecting the amount of replicating RNA and/or the virus         particles.

The p7 peptide features two transmembrane domains (TM1 and TM2), and p7 monomers multimerize to form a putative ion channel. Additionally p7 has been shown to contain genotype specific sequences required for genotype specific interactions between p7 and other HCV proteins. Hence, new compounds targeting the putative p7 ion-channel and autoprotease inhibitors interfering with NS2, and drugs targeting cellular proteins involved in the described processes can be tested.

The inventors of the present invention showed that JFH1-based viruses can be used for testing putative anti-HCV antivirals. Huh7.5 cultures infected with JFH1-based recombinants of genotypes 1a, 1b, 2a, 2b, and 6a were treated with 500 IU/mL interferon-α2b (FIG. 3A, B), 20 μM ribavirin (FIG. 3C, D) or 50 μM amantadine (FIG. 3E, F), respectively. A combination of interferon-α2b and ribavirin is the only currently licensed treatment of HCV infected patients. While sustained viral response (SVR) can be achieved in 80-90% of genotype 2 and 3 infected patients treated with this combination therapy, SVR is only seen in 40-50% of genotype 1 and 4 infected patients. Sequence differences of several genome regions, especially E2 and NS5A, are suggested to be responsible for this differential response. The ion-channel blocker amantadine is used in treatment of influenza and has been suggested to block HCV p7. At the tested concentrations, no significant cytotoxic effect was observed. After 72 hrs of interferon-α2b treatment, an >60% decrease in the number of infected cells and a ˜2 log decrease in supernatant HCV RNA titers was observed (FIG. 3A, B). Treatment with ribavirin and amantadine had no apparent effect (FIGS. 3C-F). This is in line with previous studies, in which interferon decreased replication of J6/JFH, whereas ribavirin and amantadine did not decrease production of infectious virus in JFH1 cultures or cultures with genotype 1a (H77), 1b (Con1) or 2a (J6) JFH1-based recombinants. Genotype specific susceptibility to interferon-α2 in patients was attributed different genome regions, especially in E2 and NS5A. With the relatively high doses used for treatment of genotype 1-6 infected cultures, we did not observe any genotype specific effect; in future studies, it will be of interest to test different interferon doses and different HCV isolates, preferably from patients showing different responses to interferon. Differential sensitivity to interferon could also be mediated by the UTRs or NS3 to NS5B proteins, which are genotype 2a specific in all the recombinants tested. In conclusion, the developed systems can be applied to test the antiviral potential of known and newly developed therapeutics and to test, which HCV genome regions mediated resistance to treatment.

Thus, one embodiment of the present invention relates to a method for screening an anti-hepatitis C virus substance, comprising

-   -   a) culturing at least one selected from the group consisting of         a cell according to the present invention, a hepatitis C virus         infected cell according to the present invention and a hepatitis         C virus particle obtainable by the present invention together         with a hepatitis C virus permissive cell,     -   b) subjecting said virus or virus infected cell culture to the         anti-hepatitis C virus substance, and     -   c) detecting the replicating RNA and/or the virus particles in         the resulting culture.

In another embodiment, the inhibition of HCV replication and/or infection and/or pathogenesis includes inhibition of downstream effects of HCV. In one embodiment, downstream effects include neoplastic disease, including, in one embodiment, the development of hepatocellular carcinoma.

In one embodiment, the invention provides a method of screening for anti-HCV therapeutics, the method comprising contacting a cell with an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, comprising a chimeric HCV genome and contacting the cell with a candidate molecule, independently contacting the cell with a placebo and determining the effects of the candidate molecule on HCV infection, replication, or cell-to-cell spread, versus the effects of the placebo, wherein a decrease in the level of HCV infection, replication, or cell-to-cell spread indicates the candidate molecule is an anti-HCV therapeutic.

In one embodiment, the method may be conducted be in vitro or in vivo. In one embodiment, the cells as described may be in an animal model, or a human subject, entered in a clinical trial to evaluate the efficacy of a candidate molecule. In one embodiment, the molecule is labelled for easier detection, including radio-labelled, antibody labelled for fluorescently labelled molecules, which may be detected by any means well known to one skilled in the art.

In one embodiment, the candidate molecule is an antibody.

In one embodiment, the term “antibody” refers to intact molecules as well as functional fragments thereof, such as Fab, F(ab′)2, and Fv. In one embodiment, the term “Fab” refers to a fragment, which contains a monovalent antigen-binding fragment of an antibody molecule, and in one embodiment, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain, or in another embodiment can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. In one embodiment, the term “F(ab′)2”, refers to the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds. In another embodiment, the term “Fv” refers to a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains, and in another embodiment, the term “single chain antibody” or “SCA” refers to a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of producing these fragments are known in the art.

In another embodiment, the candidate molecule is a small molecule. In one embodiment, the phrase “small molecule” refers to, inter-alia, synthetic organic structures typical of pharmaceuticals, peptides, nucleic acids, peptide nucleic acids, carbohydrates, lipids, and others, as will be appreciated by one skilled in the art. In another embodiment, small molecules, may refer to chemically synthesized peptidomimetics of the 6-mer to 9-mer peptides of the invention.

In another embodiment, the candidate molecule is a nucleic acid. Numerous nucleic acid molecules can be envisioned for use in such applications, including antisense, siRNA, ribozymes, etc., as will be appreciated by one skilled in the art.

It is to be understood that the candidate molecule identified and/or evaluated by the methods of this invention, may be any compound, including, inter-alia, a crystal, protein, peptide or nucleic acid, and may comprise an HCV viral product or derivative thereof, of a cellular product or derivative thereof. The candidate molecule in other embodiments, may be isolated, generated synthetically, obtained via translation of sequences subjected to any mutagenesis technique, or obtained via protein evolution techniques, well known to those skilled in the art, each of which represents an embodiment of this invention, and may be used in the methods of this invention, as well.

In one embodiment, the compound identified in the screening methods as described, may be identified by computer modeling techniques, and others, as described herein. Verification of the activity of these compounds may be accomplished by the methods described herein, where, in one embodiment, the test compound demonstrably affects HCV infection, replication and/or pathogenesis in an assay, as described. In one embodiment, the assay is a cell-based assay, which, in one embodiment, makes use of primary isolates, or in another embodiment, cell lines, etc. In one embodiment, the cell is within a homogenate, or in another embodiment, a tissue slice, or in another embodiment, an organ culture. In one embodiment, the cell or tissue is hepatic in origin, or is a derivative thereof. In another embodiment, the cell is a commonly used mammalian cell line, which has been engineered to express key molecules known to be, or in another embodiment, thought to be involved in HCV infection, replication and/or pathogenesis.

In another embodiment, protein, or in another embodiment, peptide or in another embodiment, other inhibitors of the present invention cause inhibition of infection, replication, or pathogenesis of HCV in vitro or, in another embodiment, in vivo when introduced into a host cell containing the virus, and may exhibit, in another embodiment, an IC50 in the range of from about 0.0001 nM to 100 μM in an in vitro assay for at least one step in infection, replication, or pathogenesis of HCV, more preferably from about 0.0001 nM to 75 μM, more preferably from about 0.0001 nM to 50 μM, more preferably from about 0.0001 nM to 25 μM, more preferably from about 0.0001 nM to 10 μM, and even more preferably from about 0.0001 nM to 1 μM.

In another embodiment, the inhibitors of HCV infection, or in another embodiment, replication, or in another embodiment, pathogenesis, may be used, in another embodiment, in ex vivo scenarios, such as, for example, in routine treatment of blood products wherein a possibility of HCV infection exists, when serology shows a lack of HCV infection.

In another embodiment, the anti-HCV therapeutic compounds identified via any of the methods of the present invention can be further characterized using secondary screens in cell cultures and/or susceptible animal models. In one embodiment, a small animal model may be used, such as, for example, a tree shrew Tupaia belangeri chinensis. In another embodiment, an animal model may make use of a chimpanzee. Test animals may be treated with the candidate compounds that produced the strongest inhibitory effects in any of the assays/methods of this invention. In another embodiment, the animal models provide a platform for pharmacokinetic and toxicology studies.

Vaccines

The construct according to the invention by itself can also be used for various purposes in all its embodiments. This includes the construction of hepatitis C viruses or HCV-like particles and their production in cell cultures as described.

These HCV or HCV-like particles can be used in particular as vaccine. Thus, one embodiment of the present invention relates to a hepatitis C vaccine comprising a hepatitis C virus particle according to the invention or a part thereof.

In another embodiment, the nucleic acids, vectors, viruses, or viral particles may be further engineered to express a heterologous protein, which, in another embodiment, is mammalian or a derivative thereof, which is useful in combating HCV infection or disease progression. Such proteins may comprise cytokines, growth factors, tumor suppressors, or in one embodiment, may following infection, be expressed predominantly or exclusively on an infected cell surface. According to this aspect of the invention, and in one embodiment, such molecules may include costimulatory molecules, which may serve to enhance immune response to infected cells, or preneoplastic cells, or neoplastic cells, which may have become preneoplastic or neoplastic as a result of HCV infection. In one embodiment, the heterologous sequence encoded in the nucleic acids, vectors, viruses, or viral particles of this invention may be involved in enhanced uptake of a nucleic acids, vectors, viruses, or viral particles, and may specifically target receptors thought to mediate HCV infection.

Further, the present invention relates to a method for producing a hepatitis C virus vaccine comprising using a hepatitis C virus particle according to the invention as an antigen, and naturally any antibody against such hepatitis C virus particle.

Uses

The genotype 2b cell culture system developed of the present invention will be a valuable tool to address different research topics. It will allow the genotype specific study of functions of the structural proteins (Core, E1, E2) as well as p7 and NS2 using reverse genetics. While the replicon systems facilitated testing of drugs interfering with replication such as NS3/4A protease and polymerase inhibitors, the system developed in this study is ideal for the genotype 2b specific testing of new drugs, such as drugs interfering with viral entry, such as fusion inhibitors, as well as assembly and release.

Accordingly the genotype 2b developed cell culture systems allows individual patient targeting. This means that when a new potential therapeutic candidate is discovered it is possible to test this particular candidate or combination of candidates on this genotype. Knowing which specific genotype the candidate is functioning towards, it allows an individual treatment of each patient dependent on which specific genotype the patient is infected with. Furthermore these cell culture systems allow the development of antibodies and vaccines targeting individual patients.

In addition new therapeutics targeting the putative p7 ion-channel and protease inhibitors targeting NS2 can be tested specifically for genotype 2b thus allowing individual patient targeting.

J8/JFH1 recombinant viruses will be well suited for screenings for broadly reactive neutralizing antibodies, thus aiding vaccine development.

The replication level of a virus can be determined, in other embodiments, using techniques known in the art, and in other embodiments, as exemplified herein. For example, the genome level can be determined using RT-PCR. To determine the level of a viral protein, one can use techniques including ELISA, immunoprecipitation, immunofluorescence, EIA, RIA, and Western blotting analysis. To determine the replication rate of a virus, one can use the method described in, e.g., Billaud et al., Virology 266 (2000) 180-188.

In one embodiment, the invention provides a method of identifying sequences in HCV associated with HCV pathogenicity, comprising contacting cells with an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, comprising a chimeric HCV genome, contacting cells with an isolated nucleic acid molecule comprising at least one mutation of the chimeric HCV genome, independently culturing the cells and determining HCV infection, replication, or cell-to-cell spread, in cells contacted with the mutant, versus the chimeric HCV, whereby changes in HCV infection, replication, or cell-to-cell spread in cells contacted with the mutant virus shows the mutation is in an HCV sequence associated with HCV pathogenicity.

In one embodiment, the invention provides a method of identifying HCV variants with improved growth in cell culture, the method comprising contacting cells with an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, comprising a chimeric HCV genome contacting cells with an isolated nucleic acid molecule comprising or not comprising mutations of the chimeric HCV genome, independently culturing the cells and determining HCV infection, replication, or cell-to-cell spread, in cells contacted with the chimeric HCV or the mutated virus, whereby enhanced HCV infection, replication, or cell-to-cell spread in cells contacted with the mutated virus shows that the HCV variant has improved growth in cell culture. In some embodiments, HCV variants are selected for enhanced replication, over a long course of time, in vitro culture systems. According to this aspect of the invention, and in some embodiments, cells contacted with the variants are characterized by reduced infection, as compared to cells contacted with the chimeric HCV.

The developed systems can be used to quantify HCV proteins as well as their influence on and interaction with host cell factors. In the present invention, the inventors used confocal microscopy based image analysis to quantify HCV Core and NS5A protein, the amount of intracellular lipids and interaction of Core and NS5A with intracellular lipids. The inventors evaluated spread of in vitro HCV infection by quantitative confocal microscopy based imaging. In a blinded study, increasing amounts of Core and NS5A relative to the number of total cells were detected during days 3-10 for genotype 1, 2 and 6 recombinants (FIG. 2C, D), suggesting that this methodology could be an effective tool to evaluate HCV infection in vitro. The method also readily detected a nonspecific background staining with the anti-Core antibody, whereas the anti-NS5A gave no such signal. Thus, for optimization this quantification method requires attention to the selection of antibodies for immunostaining.

The HCV lifecycle depends on the lipid metabolism and Core has been suspected to induce hepatocellular steatosis in genotype 3 patients. In the present invention, big variation in the lipid content of non-infected Huh7.5 cells was found; during 10 days, infection with genotype 1, 2 and 6 recombinants did not induce intracellular lipid accumulation (FIG. 5), and no genotype specific differences in lipid content were found at peak infection (Table 4). A short-term infection in cell culture might not induce the changes in lipid metabolism leading to steatosis in chronically infected patients. Even though the inventors analyzed an average of 660 cells per culture for each time-point, it is evident that there was variation in the lipid content in infected as well as non-infected cells, which might mask possible subtle differences in lipid content induced by HCV. Furthermore, the inventors based their analysis on quantification of fluorescent intensity to quantify the total amount of lipids in the cell cytoplasm. Thus, morphological differences of lipid droplets between infected and non-infected cells were not analysed, which has been carried out in HCV infected cells by electron microscopy and in HCV Core expressing cells by confocal microscopy.

The present inventors found HCV Core to co-localize with lipid droplets for genotype 1, 2 and 6 recombinants (FIG. 6) as described by others for genotype 2a; further, co-localization of NS5A with lipid droplets was detected for genotype 1, 2 and 6 recombinants (FIG. 6), indicating either a direct or Core-mediated association. Interestingly, the interaction of NS5A with Core was found to play an important role in regulating the early phase of HCV particle formation.

Kits

In a related aspect, the invention also provides a test kit for HCV comprising HCV virus components, and a diagnostic test kit for HCV comprising components derived from an HCV virus as described herein.

Furthermore the invention also provide test kits, for screening for new HCV genotype 2b inhibitors, neutralizing and cross neutralizing antibodies, comprising HCV virus components.

General

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

As will be apparent, preferred features and characteristics of one aspect of the invention may be applicable to other aspects of the invention. The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus showed be the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced by reference therein.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In addition, singular reference does not exclude a plurality. Thus, references to “a”, “an”, “first”, “second” etc. do not preclude a plurality.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus showed be the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced by reference therein.

The invention will hereinafter be described by way of the following non-limiting Figures and Examples.

Sequences SEQ ID NO DNA/amino acid (AA) Name SEQ ID NO: 1 DNA J8/JFH1 SEQ ID NO: 2 AA J8/JFH1 SEQ ID NO: 3 DNA -84S_HCV-MOD SEQ ID NO: 4 DNA J8 R1458 fus SEQ ID NO: 5 DNA J8 F1286 fus SEQ ID NO: 6 DNA J8R2309 SEQ ID NO: 7 DNA J8F1989 SEQ ID NO: 8 DNA J8 R3003 SEQ ID NO: 9 DNA J8F2762 SEQ ID NO: 10 DNA 3774R_J6 SEQ ID NO: 11 DNA J8F3167 SEQ ID NO: 12 DNA 4118R_JFH1 SEQ ID NO: 13 DNA J8/JFH1(G4458A) SEQ ID NO: 14 AA J8/JFH1(G4458A)

EXAMPLES Materials and Methods

Source of HCV Genotype 2b

A plasma pool of strain J8 was prepared from acute-phase plasmapheresis units collected from an experimentally infected chimpanzee. This plasma pool has an HCV RNA titer of approximately 10^(4.2) IU/ml and an infectivity titer of approximately 10⁴ chimpanzee infectious doses/ml (Engle et al. 2008).

Construction of J8/JFH1 Intergenotypic Recombinant; RNA Extraction, RT-PCR, PCR and Cloning

RNA was extracted from plasma and cell culture supernatant using either the High Pure Viral Nucleic Acid Kit (Roche) or the TRIzol (Invitrogen) procedure according to manufacturers protocol. All reverse transcription-polymerase chain reactions (RT-PCR) were carried out using RNA extracted from 100 μL plasma or cell culture supernatant. Primers (TAG Copenhagen) were 1.25 μM and dNTPs (Invitrogen) were 0.5 mM in RT reactions. For denaturation, RNA was incubated for 2′ at 65° C. together with primer and dNTPs and placed on ice. cDNA syntheses was done in a 20 μL volume with enzyme and incubation times as described. The final RT reaction was treated with 1-4 U RNase H (Invitrogen) and 1000 U RNase T1 (Ambion) for 20′ at 37° C. to degrade RNA. All PCR reactions were done using 2.5 μL cDNA reaction as template in a 50 μL reaction volume. Final concentrations of primer and dNTPs in PCR were 0.2 μM and 0.25 mM respectively. PCR fragments were amplified from the J8 plasma pool with Advantage 2 PCR system (Clontech) and subcloned (Invitrogen, TA-TOPO cloning kits). The J8 Core-NS2 consensus sequence was determined from 4-7 clones. pJ8/JFH1 was constructed by insertion of the EcoRI-AvrII fragment of a fusion product containing JFH1 5′UTR (including EcoRI in pFL-J6/JFH upstream of the HCV sequence), the consensus Core-NS2 sequence of J8, and JFH1 NS3 into pFL-J6/JFH. JFH1 fragments were amplified from pFL-J6/JFH. J8 fragment was amplified from clones derived from the respective plasma pool. Amplification of PCR products for fusion PCR and fusion PCRs were carried out with Pfu Polymerase (Stratagene). Restriction enzymes were purchased from New England Biolabs and ligations were performed using Rapid DNA ligation kit (Roche) according to the protocol. TOP10 chemically competent bacteria (Invitrogen) were used for all bacterial cloning according to the protocol. DNA preparations were carried out using QIAGEN QIAprep spin miniprep kit or QIAfilter plasmid maxi kit (both Qiagen).

In Vitro Transcription

For in vitro transcription 5 μg plasmid was XbaI-linearized (New England Biolabs). Transcription was carried out for 2 hrs with T7 RNA polymerase (Promega) according to protocol. RNA production was evaluated by gel electrophoresis.

Huh7.5 Cell Culture and Generation of Virus Stocks

The human hepatoma cell line Huh7.5 is an INF-α cured clone of the Huh7 hepatoma cell line, with increased HCV replication abilities. Cells were cultured in D-MEM+4500 mg/L Glucose+GlutaMAX-I+Pyruvate (Invitrogen) containing 10% heat inactivated fetal bovine serum (FBS) (Sigma), penicillin at 100 units/ml and streptomycin at 100 mg/ml (Invitrogen) at 5% CO2 and 37° C. Every 2-3 days cells were split after washing with PBS and trypsinizing (Trypsin/EDTA, Invitrogen). Supernatants were sterile filtered to exclude cells and debris and stored at −80° C.

For transfection of HCV RNA transcripts, naïve Huh7.5 cells were plated at 4×10⁵/well in 6-well plates the day before transfection. Prior to transfection 2.5 μg of unpurified RNA transcripts were incubated with Lipofectamine2000 (Invitrogen) in 500 μL Opti-MEM (Invitrogen) for 20′ at room temperature. RNA-Lipofectamine2000 transfection complexes were left on cells for 12-24 hrs before washing.

To prove the production of infectious viruses, sterile filtered supernatant from infected cultures was used to infect naïve Huh7.5 cells. Unless other is described, 1 mL supernatant was used for infection of Huh7.5 cells plated in 6-well plates at 4×10⁵/well the day before. Supernatants were left on cells for 3-8 hrs as described in figure and table legends.

Negative controls in transfections were RNA transcripts from replication deficient JFH1-based genomes (with the GND motif); in the kinetic experiment, non-infected cells were used (data not shown).

Viral spread was monitored by HCV Core or NS5A immunostainings with mouse anti-HCV core protein monoclonal antibody (B2) (Anogen, Yes Biotech Laboratories) or anti-NS5A, 9E10, respectively, as described in the following section.

Supernatant infectivity titers were determined as 50% tissue culture infectious dose (TCID₅₀)/mL or as focus forming units (FFU)/mL, as described in the following section.

Supernatant HCV RNA titers were measured by a 5′ UTR based Real Time RT-PCR as described below.

For analysis of intracellular infectivity and HCV RNA titers, 1×10⁵ cells were pelleted by centrifugation at 500 g for 5 min at 4° C. Pellets were resuspended in complete culture medium and subjected to four freeze/thaw cycles in liquid nitrogen/37° C.; medium was clarified by 2× centrifugation at 1500 g for 5 min and analysed as above.

For generation of virus stocks, Huh7.5 cells were infected at a multiplicity of infection (MOI) of ˜0.003. After viral spread to >80% of the culture (Core or NS5A immunostaining), supernatants were filtered, aliquoted and stored at −80° C. Size of each viral stock was ˜100 mL.

Immunostainings for HCV Antigens and Lipids; Titration of Infectivity

For staining, cells grown over night on 4- or 8-well chamberslides (Nunc) were washed 2× with PBS and fixed for 5 minutes with acetone. After washing 2× with PBS and 1× with PBS/Tween-20 (0.1%), slides were incubated with 1° antibody (MAB Murine Anti-Human HCV, Core Protein, Clone B2 (Anogen) or anti-NS5A, 9E10 (gift from C. Rice, Rockefeller University) used at 1:200 in PBS containing 5% bovine serum albumine (BSA) for 20′ at room temperature. After washing as above, 2° antibody (Alexa Fluor 594 goat anti-mouse IgG (H+L)) and Hoechst33342 (both Invitrogen) for cell nuclei counterstaining, used at 1:500 and 1:10000 dilutions, respectively in PBS/Tween, was added for 5 min. Lipids were stained with oil red O (Fisher scientific) as described previously. Finally, slides were washed with PBS, mounted with Fluoromount-G (Southern Biotech) and cover slipped. Staining was visualized using a Leica TCS SP5 confocal microscope. Percentage of infected cells was evaluated by assigning values of 0% (no cells infected), 1% (or below), 5%, 10-90% in steps of 10, 95% and 100% (all cells infected).

Viral infectivity titers were determined by the tissue culture infectious dose 50 (TCID₅₀) or focus forming unit method. 6×10³/well naive Huh7.5 cells were plated out in a poly-D-lysine coated 96-well plate (Nunc) the day before infection. Cells were then incubated with 10-fold dilutions of cell culture supernatants. For TCID₅₀ determinations, 6 replicates per dilution were incubated for 2-3 days. For FFU determinations, wells were incubated for 48 rs. After incubation, cells were permeabilized for 5′ with cold methanol. After washing 1× with PBS and 1× with PBS/Tween-20, blocking was carried out for 20′ with sterile filtered 1% BSA/0.2% skim milk in PBS followed by a 5′ blocking of endogenous peroxidase activity using 3% H2O2. Cells were washed as above and incubated with a 1:200 dilution of 1° Ab α-NS5A (9E10) in PBS/0.1% tween-20 over night at 4° C. After washing, a 1:300 dilution of 2° Ab HRP-goat anti-mouse IgG (H+L) (Amersham Biosciences) in PBS/0.1% tween-20 was added and incubated for 30′ at room temperature. Staining was developed using DAB substrate kit (DAKO) for 30′ after washing. In TCID₅₀ determinations, wells were scored positive if one or more cells were infected, and the TCID₅₀ was calculated according to the Reed and Muench method. TCID₅₀ values are derived from single or multiple determinations as indicated. FFU determinations are based on counts of wells with 5-100 FFU and three independent virus dilutions with one replicate each. However, FFU calculations for virus stocks (Table 3) were based on two independent virus dilutions with 6 replicates each.

Real-Time PCR (TaqMan) Assay for Determination of HCV RNA Titers.

Supernatant HCV RNA titers were measured by a 5′ UTR based Real Time RT-PCR. RNA was purified from 200 μL of heat inactivated (56° C. for 30′) cell culture supernatant and eluted in a final volume of 50 μL using the Total Nucleic Acid Isolation Kit (Roche) in combination with the Total NA Variable Elution Volume protocol on a MagNA Pure LC Instrument (Roche). As an internal control, Phocine Distemper Virus (PDV) was added to the lysis buffer in a concentration titrated to yield a Ct of ˜32 upon real-time PCR analysis. In parallel to RNA purified from cell culture supernatants a quantitative HCV standard panel covering RNA concentrations of 0 to 5×106 IU/mL in one-log increments (OptiQuant HCV Panel, AcroMetrix) was analysed. Real-time PCR analyses of HCV and PDV RNA were carried out in two separate reactions using the TaqMan EZ RT-PCR Kit (Applied Biosystems). For HCV, primers and a FAM-labelled MGB-probe were directed against the 5′ UTR and were previously shown to perform equivalently against a panel of the six major HCV genotypes in a different TaqMan assay (Engle et al. 2008). For PDV, a ready-to-use primer/probe mix was used (Dr. H. G. M. Niesters, Department of Virology, Erasmus Medical Centre, Rotterdam, The Netherlands). The PCR analysis was performed on a 7500 Real-Time PCR System (Applied Biosystems) using 50° C. for 2′, 60° C. for 30′ and 95° C. for 5′ followed by 45 cycles of 94° C. for 20″ and 62° C. for 1′. HCV RNA titers (IU/ml) were calculated using a standard curve created from the known concentrations of the standard panel and their corresponding Ct values. The reproducible detection limit of the assay was 500 IU/ml. In order to confirm successful purification, amplification and the absence of PCR inhibitors, the Ct value of the PDV reaction was compared to the expected Ct value (based on a mean of all previous runs; n>9) using the MedLab QC freeware programme. The results of samples with an actual Ct value within ±2SD of the expected Ct value were accepted.

Confocal Imaging and Quantification

For evaluation of intracellular lipid and HCV antigen content, 5×10⁴ cells were co-stained with oil red O and anti-HCV Core or NS5A. Image stacks, each comprised of four sections, were collected on a TCS-SP2 confocal microscope (Leica) through either a 40×PL APO (Na=1.25) or a 63×PL APO (Na=1.40) objective for quantification of fluorescence intensity or analysis of co-localization, respectively. Scanning was performed sequentially in 1024×1024 format with a step size of 0.48 μm, with identical settings and avoidance of pixel saturation. The intensity of the oil red O and HCV antigen signals of 6 and 3 image stacks (each containing 110 cells on average), respectively, were quantified per culture using Imaris 6.1.0 software (Bitplane). Fluorescent signals of oil red O and HCV antigen staining was rendered in 3D by constructing an isosurface and subsequently quantifying the total voxel intensity. Quantifications were expressed relative to the number of Hoechst stained nuclei per image stack. For analysis of the percentage of HCV antigen co-localized with lipid (oil red O), thresholds for each channel were set to exclude any background noise. A contour surface was first modulated on regions without cells allowing for masking of channels to be analyzed. At least 20 cells were analyzed per culture. Throughout, all scannings and subsequent image analyses were performed blinded (under code) (see also FIGS. 5-6).

Treatment, Receptor Blocking and Neutralization

For treatment, interferon-α2b (Schering-Plough), ribavirin (Sigma) or amantadine (Sigma) was used; cell viability was monitored with CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega).

For blocking of CD81 and SR-BI and neutralization assays, Huh7.5 cells were plated 6×10³ per well of a poly-D-lysine-coated 96-well plate and incubated for 24 hrs. For blocking experiments, cells were incubated with anti-CD81 (JS-81; BD Biosciences Pharmingen, Franklin Lakes, N.J.) or isotypematched control antibody (anti-human immunodeficiency virus, p24, clone Kal-1; DAKO) and rabbit polyclonal anti-SR-BI (GeneTex) or rabbit polyclonal control antibody (anti-human Retinoblastoma (Rb) Ab-6, Thermo Scientific), respectively, for 1 hr. Subsequently, cells were infected with ˜150 FFU of the respective virus for 3 hrs followed by washing with PBS. After 48 hrs of incubation with normal growth medium, cells were stained for HCV NS5A to determine the number of focus forming units (FFU) per well. Experiments were performed in triplicates unless stated otherwise. Percent inhibition by anti-CD81 and anti-SR-BI was calculated by comparison to the FFU mean of at least 3 replicate wells incubated with virus only.

For neutralization, heat inactivated sera were pre-incubated with ˜30-150 FFU for 1 hr at 37° C., preceding 3 hrs incubation on 6×10³ Huh7.5 cells. After 48 hrs incubation with normal growth medium, cultures were immunostained for NS5A, and the number of FFU was determined. Neutralization experiments were performed in triplicates and percent inhibition by patient sera was calculated by comparison to the FFU mean of at least 3 replicate wells incubated with virus only. Sera used for neutralization were derived for from persistently infected Patient H (2006, year 29 after infection, genotype 1a), an Egyptian Patient (AA, 1994, genotype 4a), and a South African hepatocellular carcinoma patient (SA3, genotype 5a).

Sequencing of Cell Culture Derived HCV

Direct sequencing of complete ORF was done to identify adaptive mutations. RNA extraction and reverse transcription was done as described above using 400 U SuperScriptIII (Invitrogen) and RT-primer. 1st round PCR was performed in a 50 μL volume on 2.5 μL of the cDNA reaction using the Advantage 2 PCR Enzyme System (Clontech). Cycle parameters were 5 cycles of 35″ at 99° C., 30″ at 67° C. and 10′ at 68° C., 10 cycles of 35″ at 99° C., 30″ at 67° C. and 11′ at 68° C., 10 cycles of 35″ at 99° C., 30″ at 67° C. and 12′ at 68° C. and 10 cycles of 35″ at 99° C., 30″ at 67° C. and 13′ at 68° C. 12˜1 kb products were synthesized in a nested PCR covering the entire ORF Table 1). PCR was set up as above using 2.5 μL of the 1st round PCR for each reaction. Initial denaturation was 35 sec at 99° C. followed by 35 cycles with 35 sec at 99° C., 30 sec at 67° C. and 6 min at 68° C. Genotype 2b specific primers used in 2^(nd) round of the long RT-PCR procedure are given in Table 1. PCR products were agarose gel purified and directly sequenced in both directions.

Sequencing, Sequence Analysis and Databases

All sequence reactions was performed at Macrogen Inc., Seoul, South Korea. Sequence analysis was performed with Sequencher 4.7, Gene Codes Corporation and freeware BioEdit v. 7.0.5. HCV sequences used for alignments were retrieved from The European HCV database (euHCVdb; euhcvdb.ibcp.fr/euHCVdb/) and the American HCV database (LANL; hcv.lanl.gov/content/hcv-db/index).

Example 1 Cell Culture Adaptation of Intergenotypic 2b/2a Recombinant (J8/JFH1)

In two independent RNA transfections of Huh7.5 cells, J8/JFH1 spread comparably to J6/JFH with approximately 20% and 80% HCV antigen positive cells on days 1 and 4, respectively (FIG. 1A). The supernatant infectivity titers increased from ˜10^(1.5) TCID₅₀/mL on day 1 to ˜10^(3.5) TCID₅₀/mL on day 4 (FIG. 1B). During viral passage (FIG. 1C), HCV RNA and infectivity titers were ˜10⁷ IU/mL (FIG. 1D) and ˜10^(4.5) TCID₅₀/mL (data not shown), respectively. Analysis of recovered viruses (days 10 and 13 post-infection, respectively) showed that J8/JFH1 was genetically stable in two first passages performed from the first transfection (FIGS. 1C and D). In the passage of virus from the second transfection (FIGS. 1C and D), G4458A (R1373Q) was present as a 50/50 quasispecies with the original sequence (virus from day 13 post-infection).

Example 2 Titrated Stocks of Genotype 1, 2 and 6 Viruses

The supernatant virus stocks of the JFH1-based intergenotypic recombinants, as well as J6/JFH were characterized (Table 3). Infectivity titers ranged from 10^(3.7) to 10^(5.2) TCID₅₀/mL and HCV RNA titers ranged from 10^(7.0) to 10^(7.6) IU/mL with specific infectivities (defined as infectious titer relative to the HCV RNA titer) of 1/251-1/3901 TCID₅₀/IU.

There was a good correlation between the infectivity titers determined as TCID₅₀/mL and FFU/mL, respectively (Table 3).

Example 3 Comparative Kinetic Studies of Intergenotypic Viruses

To further characterize the different genotype viruses, a kinetic experiment with genotype 1, 2 and 6 recombinants was performed. After inoculation of Huh7.5 cultures with the respective stock viruses at an MOI of 0.003, efficient spread in 5-10 days (FIG. 2A) was observed, paralleled by a 2-3 log increase in HCV RNA titers to peak titers of >10⁷ IU/mL (FIG. 2B). In addition, an increase in intracellular Core and NS5A antigen by microscopy based image analyses was observed (FIG. 2C, D). In contrast, increasing infection with genotypes 1-6 did not lead to a change in the average lipid content per cell that was greater than the range of natural variation observed for non-infected cultures (FIG. 5). At peak infection (defined as the first time point with supernatant HCV RNA titers 10⁷ IU/mL), intracellular HCV RNA and intra- and extracellular infectivity titers were measured (Table 4). Intracellular specific infectivity was at least one order of magnitude lower than extracellular specific infectivity for the respective viruses. It was found that genotype 1, 2 and 6 cultures did not show significant differences in the average intracellular lipid content at the peak of infection (Table 4).

By standard confocal microscopy of genotype 1, 2 and 6 infected Huh7.5 cells co-localization of lipid droplets with Core and NS5A, respectively was observed (data not shown). Using confocal microscopy based image analyses the degree of this co-localization was determined. The analysis indicated that under the set conditions (see Materials and Methods) 5-15% of HCV Core co-localized with lipid droplets with no genotype specific differences. In addition, 10-20% of HCV NS5A co-localized with lipid droplets (FIG. 6).

Example 4 Treatment with Interferon, Ribavirin and Amantadine

The therapeutic potential of drugs currently used or tested for treatment of HCV on Huh7.5 cultures infected with genotype 1, 2 and 6 recombinants was studied. Cells obtained on day 5 of the kinetic experiment (FIG. 2A) were treated with 500 IU/mL interferon-α2b (FIG. 3A, B), 20 μM ribavirin (FIG. 3C, D) or 50 μM amantadine (FIG. 3E, F), respectively. No significant cytotoxic effect was observed (data not shown). After 72 hrs interferon-α2b treatment, an at least 60% decrease in the number of infected cells and a ˜2 log decrease in supernatant HCV RNA titers was observed (FIG. 3A, B). Treatment with ribavirin and amantadine did not decrease the number of infected cells or supernatant HCV RNA titers (FIGS. 3C-F). Thus, only interferon-α2b had an antiviral effect with no major differences regarding the genotype of the JFH1-based recombinants treated.

Example 5 Importance of CD81 and SR-B1 for HCV Genotype 1, 2 and 6 Infection

Infection with genotype 1, 2 and 6 recombinants was blocked by anti-CD81 in a dose dependent manner (FIG. 4A); >90% inhibition was observed at 2.5 μg/mL anti-CD81, whereas at 0.02 and/or 0.004 μg/mL anti-CD81<50% inhibition was found. In SR-BI blocking experiments, the present inventors showed for all genotype recombinants >90% infection inhibition with a 1:10 dilution of polyclonal anti-SR-BI (FIG. 4B). This inhibition was dose dependent, and at a 1:640 dilution <50% inhibition was found for all genotype recombinants.

Example 6 Cross-Genotype Neutralization with Chronic Phase HCV Patient Sera

Chronic phase sera from patients infected with genotypes 1a (H06), 4a (AA) and 5a (SA3) with relative high neutralization titers against the homologous genotype virus was identified. The cross-genotype neutralization potential of these sera against 1a, 2a, 3a, 4a, 5a and 6a viruses were tested previously (Table 2). These sera also showed high 50% neutralization titers against the 7a virus (Table 2). Relative high neutralization titers were found against 1b and 2b viruses with the H06 sera, whereas the AA and SA3 sera showed limited neutralization of these viruses (Table 2). It is of interest, that different subtypes as 2a and 2b show a differential susceptibility to neutralization.

FIGURE LEGENDS

FIG. 1

Viability of J8/JFH1 recombinant in Huh7.5 cells. (A and B) Transfection experiment. Huh7.5 cells were transfected in parallel with RNA transcripts from pJ8/JFH1 (two separate experiments; exp. 1 and exp. 2), pJ6/JFH (positive control), pJ6/JFH (GND) (negative control). (A) After immunostaining, the percentage of HCV Core positive cells was scored by fluorescence microscopy. (B) Infectivity titers of transfection supernatants were measured as TCID₅₀/mL in single determinations. (C and D) First viral passage. Naïve Huh7.5 cultures were inoculated for 4 hrs with supernatant derived on day 4 after transfection with J8/JFH1 (exp. 1 and exp. 2) and J6/JFH RNA transcripts (A, B) at an MOI of 0.006. As negative control, supernatant derived at day 4 after transfection with S52/JFH1(GND) was used. (C) After immunostaining, the percentage of HCV Core positive cells was scored by fluorescence microscopy. (D) Supernatant HCV RNA titers were measured by Real-Time RT-PCR.

FIG. 2

Comparative kinetics studies of intergenotypic viruses of genotypes 1, 2 and 6. Huh7.5 cells were inoculated with the respective stock virus (Table 3) for 6 hrs (MOI 0.003); J4/JFH1_(F886L,Q21496L) was from a different virus stock. (A) After immunostaining, the percentage of HCV NS5A positive cells was scored by fluorescence microscopy. (B) Supernatant HCV RNA titers were measured by Real-Time RT-PCR. (C, D) Average content of intracellular HCV Core and NS5A was determined by confocal microscopy based quantitative image analysis after immunostaining for the respective antigen. For each culture and antigen, 3 image stacks were acquired, each comprising an average of 110 cells. Average content of HCV antigen per cell was determined for each image using Imaris 6.1.0 software. Means of the 3 datasets are shown. AU, arbitrary units. None-infected negative control cells are not shown; for Core stainings, a background signal of 2.5 Log₁₀ AU (mean of 12 determinations) was recorded, whereas NS5A did not show a background signal.

FIG. 3

Treatment of intergenotypic viruses of genotype 1, 2 and 6 with putative antivirals. 4×10⁵ Huh7.5 cells, derived on day 5 of the kinetic experiment (FIG. 2), were plated in 6 well dishes (−12 hrs). After 12 hrs, cell were treated at 0, 6, 12, 24, 48 and 72 hrs with 500 IU/mL interferon-α2b (A, B), 20 μM ribavirin (C, D) or 50 μM amantadine (E, F), respectively. At the indicated time points, percentage of HCV NS5A positive cells was determined using immunostaining and fluorescence microscopy (A, C, E); supernatant HCV RNA titers were measured by Real-Time RT-PCR (B, D, F).

FIG. 4

Importance of CD81 and SR-BI for entry of intergenotypic viruses. 6×10³ Huh7.5 cells per well of a 96 well plate were treated for 1 hr with either anti-CD81 (A) or anti-SR-BI (B) at the indicated concentrations. ˜150 FFU of the respective virus were added for 3 hrs. Virus stocks shown in Table 3 were used. After 48 hrs, the number of FFU was evaluated following immunostaining for HCV NS5A. % inhibition was calculated by relating the number of FFU/well to the mean number of FFU/well of 3 untreated wells. Means of triplicates and standard errors of the mean are shown. Control antibody preparations specified in Materials and Methods did not show any inhibitory effect at the equivalent concentrations. Stars, value<0. Data shown in B were generated in three different experiments (1st experiment: 1:10, 1:40 and 1:160 dilutions (1:160 not shown); 2nd experiment: 1:160 and 1:640 dilutions; 3rd experiment: all dilutions for J4/JFH1_(F886L,Q21496L) viruses). The efficient blocking of infection of the different genotype recombinants with anti-SR-BI was confirmed in an independent experiment (data not shown). The apparent genotype specific differences seen at 1:160 dilution were only reproducible in 2 of 3 independent experiments.

FIG. 5

Average content of intracellular lipids during infection with genotype 1, 2 and 6 viruses. At day 3, 5, 7, and 10 after infection (FIG. 2) with the indicated JFH1-based recombinants, 5×10⁴ Huh7.5 cells of the respective cultures were plated on chamber slides. After 24 hrs, lipid droplets were stained with oil-red O, HCV antigen was stained with either anti-Core or anti-NS5A antibodies, and cell nuclei were stained with Hoechst reagent. For each culture, 6 image stacks were acquired, each of them comprised of on average 110 cells, using confocal microscopy imaging. Average content of lipids per cell was determined for each image using Imaris 6.1.0 software. Means and SEM of the 6 datasets are shown. AU, arbitrary units. Star, time points not analysed.

FIG. 6

Co-localization of HCV antigens with lipid droplets. At day 5 after infection (FIG. 2) with the indicated JFH1-based recombinants, 5×10⁴ Huh7.5 cells of the respective cultures were plated on chamber slides. After 24 hrs, lipid droplets were stained with oil-red O, HCV antigen was stained with either anti-Core or anti-NS5A antibodies, and cell nuclei were stained with Hoechst reagent. For each culture and antigen, 4 image stacks were acquired, each comprising >20 cells, using confocal microscopy imaging. Average % of co-localization was determined for each image stack using Imaris 6.1.0. Means and SEM of the 4 datasets are shown. Low % of co-localization of Core with lipids for the none-infected culture is due to a background signal observed in Core stainings (see also FIG. 2).

Tables

TABLE 1 Primers used for J8/JFH1 long RT-PCR procedure to generate amplicons for direct sequencing of the ORF* Amplification step and amplicon 2nd round PCR Primer name SEQ ID NO Primer sequence Amplicon 1 -84S_HCV-MOD SEQ ID NO: 3 5′-GTAGCGTTGGGTTGCGAAAGGCCTTGTGGTACTGCCTGAT-3′ J8 R1458 fus SEQ ID NO: 4 5′-CAATGACTTTGGCCCACGCTCCTTG-3′ Amplicon 2 J8 F1286 fus SEQ ID NO: 5 5′-CCATCGCATGGCATGGGACATGATGC-3′ J8R2309 SEQ ID NO: 6 5′-GCAGCGATCTCCGCGCGTGAAGT-3′ Amplicon 3 J8F1989 SEQ ID NO: 7 5′-GCTTGGTTCGGCTGCACTTGGATGAAC-3′ J8 R3003 SEQ ID NO: 8 5′-GGCGTGGGTGTAGAATGACAGCCACCC-3′ Amplicon 4 J8F2762 SEQ ID NO: 9 5′-ACCACAGCAGGCTTATGCCTTGGACG-3′ 3774R_J6 SEQ ID NO: 10 5′-GGGATGACATCAGCGTTCCGCGTGACCAG-3′ Amplicon 5 J8F3167 SEQ ID NO: 11 5′-GATCACCATAGGCAGATGGACCGGCACTTA-3′ 4118R_JFH1 SEQ ID NO: 12 5′-CGCCCGAGGCCTACCTCTTCTATATC-3′ *Primers used for cDNA synthesis, 1^(st) round PCR and 2^(nd) round PCR amplicon 6 to 12 bind to the JFH1 portion of the recombinant and are given in Supplementary Material and Methods of Gottwein et al. (2007).

TABLE 2 Cross-genotype neutralization potential of chronic phase genotype 1a, 4a and 5a serum against genotype 1-7 recombinant viruses. Core-NS2 Reciprocal 50% serum neutralizing antibody titer Genotype 1a (H06) 4a (AA) 5a (SA3) 1a  1600  <100* <100 1b  800  <100*  <100* 2a  <100*  <100** <100 2b  3200  400  200 3a  <100*  <100** <100 4a 12800 6400  200 5a 25600 3200 6400 6a 204800  25600  12800  7a 25600 3200 1600

Neutralization of genotype 1a, 2a, 3a, 4a, 5a and 6a viruses with 1a (H06), 4a (AA) and 5a (SA3) chronic phase serum was described previously. Similarly, approximately 150 FFU of J4/JFH1_(F886L,Q21496L), 80 or 150 FFU of J8/JFH1, and 30 FFU of QC69/JFH1 stock viruses were pre-incubated with 2-fold dilutions of sera in triplicates, before infection of 6×10³ Huh7.5 cells for 3 hrs. After 48 hrs incubation, the number of FFUs was determined for each culture by anti-NS5A immunostaining. 50% neutralization titers indicate the serum dilution, which led to an at least 50% reduction of FFU compared to the mean of 6 non-serum treated cultures. * 50% neutralization observed at 1:50 serum dilution; ** less than 50% neutralization observed at 1:50 serum dilution.

TABLE 3 Titrated Stocks of JFH1-based Intergenotypic Recombinants of HCV Genotype 1, 2 and 6. HCV RNA Core- HCV Infectivity titer* titer # Specific NS2 Viral LOG₁₀ LOG₁₀ LOG₁₀ infectivity** Genotype Virus † Passage TCID₅₀/mL FFU/mL IU/mL TCID₅₀/IU 1a H77C/JFH1_(V787A,Q1247L) 2nd 4.3 ± 0.0 4.3 ± 0.2 7.5 ± 0.1 1/1585 1b J4/JFH1_(F886L,Q1496L) 1st 3.7 ± 0.3 3.2 ± 0.0 7.3 ± 0.1 1/3981 2a J6/JFH1 2nd 5.2 ± 0.1 5.0 ± 0.2 7.6 ± 0.0 1/251  2b J8/JFH1 1st 4.4 ± 0.1 4.1 ± 0.1 7.4 ± 0.0 1/1000 6a HK6a/JFH1_(F350S,N417T) 1st 4.4 ± 0.2 4.0 ± 0.0 7.0 ± 0.0 1/398  † HCV recombinant with engineered adaptive mutations given as subscript. HCV ORF sequences, including the presence of specific mutations, were verified by direct sequencing of stock genomes; additionally, a 50/50 quasispecies coding mutation was revealed for H77C/JFH1 (Y361Y/H). *Measured as TCID₅₀/mL (mean of four determinations, each based on serial dilution with 6 replicates per dilution; ±SEM, standard error of the mean) and FFU/mL (mean of two determinations, each based on serial dilution with 6 replicates per dilution; ±SEM), # Measured as IU/mL (mean of two determinations, ±SEM) in a Real-Time RT-PCR assay. **Determined as HCV RNA titer (IU/mL) related to HCV infectivity titer (TCID₅₀/mL).

TABLE 4 Characterization of genotype 1, 2 and 6 kinetic cultures at peak of infection. HCV RNA HCV titer** Infectivity titer& Specific EC ‡ IC ## EC ‡ IC ## infectivity HCV Antigen $ Lipid $ Core- LOG₁₀ LOG₁₀ LOG₁₀ LOG₁₀ EC ‡ IC ## Core/ NS5A/ Lipid/ NS2 Infection# IU/ IU/ FFU/ FFU/ FFU/ FFU/ cell cell cell Genotype Virus † Day* % mL 10⁵ cells mL 10⁵ cells IU IU LOG₁₀ AU LOG₁₀ LOG₁₀ 1a H77C/JFH1_(V787A,Q1247L) 7 80 7.4 7.2 4.0 2.7 1/2512 1/31623 4.9 5.5 4.3 1b J4/JFH1_(F886L,Q1496L) 7 80 7.4 6.6 4.0 1.9 1/2512 1/50119 4.9 5.5 4.4 2a J6/JFH1 7 90 8.0 7.2 5.1 2.5 1/794 1/50119 5.5 5.7 4.0 2b J8/JFH1 7 90 7.4 7.3 4.6 2.7 1/631 1/39811 5.2 5.5 4.3 6a HK6a/JFH1_(F350S,N417T) 7 80 7.0 7.0 4.1 1.9 1/794 1/125893 5.4 5.4 4.2 None None 7 0 nd nd nd nd na na 3.0 nd 4.0 † HCV recombinant with engineered adaptive mutations used in comparative kinetic study (FIG. 4). *The first time point (day), at which HCV RNA titers in culture supernatant were ≧10⁷ IU/mL (FIG. 4B). #% infected cells scored using fluorescence microscopy (FIG. 4A). **IU/mL or IU/10⁵ cells. ‡ EC, extracellular analysis was carried out on culture supernatants. ## IC, for intracellular analysis pellets of 10⁵ cells were resuspended in growth medium and subjected to 4 freeze/thaw cycles. After centrifugation, supernatants were analysed. &FFU/mL or FFU/10⁵ cells (mean of three determinations, each based on serial dilution with 1 replicate per dilution).

REFERENCES

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The invention claimed is:
 1. A nucleic acid molecule encoding an amino acid sequence with at least 98% identity to SEQ ID NO:2, wherein said molecule is a genetically engineered human Hepatitis C virus intergenotypic recombinant that comprises the structural Core, E1, and E2 genes, the p7 gene and the non-structural NS2 gene of genotype 2b, and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B, of genotype 2a, strain JFH1.
 2. The nucleic acid molecule according to claim 1, wherein said molecule comprises the nucleic acid sequence with at least 98% to SEQ ID NO: 1 and is selected from the group consisting of double stranded DNA complementary DNA (cDNA), positive-sense cDNA, negative-sense cDNA, positive-sense RNA, negative-sense RNA, and double stranded RNA.
 3. The nucleic acid molecule according to claim 1, wherein said molecule comprises one adaptive mutation, and said adaptive mutation is a replacement of G at position 4458 of SEQ ID NO: 1 with A.
 4. The nucleic acid molecule according to claim 1, wherein said molecule comprises one adaptive mutation, and said adaptive mutation is a replacement of R at position 1373 of SEQ ID NO: 2 with Q.
 5. The nucleic acid molecule according to claim 1, wherein said molecule is capable of generating a HCV infectivity titer of 10² TCID₅₀/ml (50% tissue culture infectious doses)/ml or above following transfection and/or subsequent viral passage.
 6. The nucleic acid molecule according to claim 1, wherein said molecule encodes the amino acid sequence with at least 99% identity to SEQ ID NO:
 2. 7. An isolated cell comprising a nucleic acid molecule of claim
 1. 8. The cell according to claim 7, wherein the cell is Huh7.5.
 9. The cell according to claim 7, wherein said molecule comprises one adaptive mutation and wherein said adaptive mutation is a replacement of R at position 1373 of SEQ ID NO: 2 with Q.
 10. A method for producing a hepatitis C virus particle, comprising culturing the cell according to claim 7 to allow the cell to produce the genetically engineered human Hepatitis C virus intergenotypic recombinant.
 11. An isolated hepatitis C virus particle comprising the nucleic acid molecule according to claim
 1. 12. The nucleic acid molecule according to claim 1, wherein said molecule encodes the amino acid sequence of SEQ ID NO:
 2. 13. The nucleic acid molecule according to claim 6, wherein said molecule comprises one adaptive mutation and wherein said adaptive mutation is a replacement of R at position 1373 of SEQ ID NO: 2 with Q.
 14. The nucleic acid molecule according to claim 1, wherein said molecule comprises the nucleic acid sequence with at least 99% identity to SEQ ID NO:
 1. 15. The nucleic acid molecule according to claim 1, wherein said molecule comprises the nucleic acid sequence with of SEQ ID NO:
 1. 16. The nucleic acid molecule according to claim 14, wherein said molecule comprises one adaptive mutation, and said adaptive mutation is a replacement of G at position 4458 of SEQ ID NO: 1 with A.
 17. The nucleic acid molecule according to claim 1, wherein said genetically engineered human Hepatitis C virus intergenotypic recombinant comprises the structural Core, E1, and E2 genes, the p7 gene, and the non-structural NS2 gene of genotype 2b, strain J8, and the non-structural genes NS3, NS4A, NS4B, NS5A, and NS5B of genotype 2a, strain JFH1. 